This project focuses on two major aspects of B cell production and function in humans: virgin and memory B cell generation. The differentiation steps that hemopoietic progenitor cells must undergo to give rise to clonally diverse virgin B lymphocytes will be analyzed in these experiments with an eye toward possible clinical manipulation of B cell production. Studies are proposed to dissect the (i) precise order of B-lineage differentiation events, (ii) order of transcriptional activity of genes important in this progression and (iii) assembly, expression and function of antigen receptor units as a function of differentiation stage and age. Fresh leads suggesting that B cell production in the bone marrow can be regulated both positively and negatively via non-antigen-specific receptors (IL-7, IFNalpha/Beta and CDI9) will be explored in one series of experiments. The hypothesis that B cells can be generated by an alternative differentiation pathway, involving light chain gene rearrangement before the rearrangement of conventional heavy chain genes, will be tested. In these experiments, (i) 'surrogate' heavy chains (MC) that couple kappa light chains to the Igalpha/beta signal transduction units on two B-lineage cell lines will be isolated and characterized, (ii) the surrogate RC gene cloned, sequenced, and expressed, (iii) monoclonal antibodies produced against the surrogate HC, and (iv) these reagents used as probes to identify bone marrow cells in this pathway. Studies will be conducted to pursue preliminary data suggesting the antibody repertoire may differ in B cells generated via this alternative pathway. The phenotypic and functional characteristics of memory B cells will be examined as a function of age. In these experiments IgA1 and IgA2 B cells will be purified from adult and cord blood samples for analysis of their (i) cell surface antigenic profile and activation status, (ii) mechanism of the isotype switch, (iii) antibody repertoire, (iv) expression of developmentally-regulated genes, and (v) functional capabilities. These studies of virgin IgM B cells and their IgA1 and IgA2 memory B cell progeny should provide valuable information for the design of vaccine strategy in individuals of various ages.